Genes Involved in Sex Pheromone Biosynthesis of Ephestia Cautella, an Important Food Storage Pest, Are Determined by Transcriptome Sequencing Antony Et Al
Total Page：16
File Type：pdf, Size：1020Kb
Genes involved in sex pheromone biosynthesis of Ephestia cautella, an important food storage pest, are determined by transcriptome sequencing Antony et al. Antony et al. BMC Genomics (2015) 16:532 DOI 10.1186/s12864-015-1710-2 Antony et al. BMC Genomics (2015) 16:532 DOI 10.1186/s12864-015-1710-2 RESEARCH ARTICLE Open Access Genes involved in sex pheromone biosynthesis of Ephestia cautella, an important food storage pest, are determined by transcriptome sequencing Binu Antony1*, Alan Soffan1,2, Jernej Jakše3, Sulieman Alfaifi1, Koko D. Sutanto1, Saleh A. Aldosari1, Abdulrahman S. Aldawood2 and Arnab Pain4 Abstract Background: Insects use pheromones, chemical signals that underlie all animal behaviors, for communication and for attracting mates. Synthetic pheromones are widely used in pest control strategies because they are environmentally safe. The production of insect pheromones in transgenic plants, which could be more economical and effective in producing isomerically pure compounds, has recently been successfully demonstrated. This research requires information regarding the pheromone biosynthetic pathways and the characterization of pheromone biosynthetic enzymes (PBEs). We used Illumina sequencing to characterize the pheromone gland (PG) transcriptome of the Pyralid moth, Ephestia cautella, a destructive storage pest, to reveal putative candidate genes involved in pheromone biosynthesis, release, transport and degradation. Results: We isolated the E. cautella pheromone compound as (Z,E)-9,12-tetradecadienyl acetate, and the major pheromone precursors 16:acyl, 14:acyl, E14-16:acyl, E12-14:acyl and Z9,E12-14:acyl. Based on the abundance of precursors, two possible pheromone biosynthetic pathways are proposed. Both pathways initiate from C16:acyl-CoA, with one involving Δ14 and Δ9 desaturation to generate Z9,E12-14:acyl, and the other involving the chain shortening of C16:acyl-CoA to C14:acyl-CoA, followed by Δ12 and Δ9 desaturation to generate Z9,E12-14:acyl-CoA. Then, a final reduction and acetylation generates Z9,E12-14:OAc. Illumina sequencing yielded 83,792 transcripts, and we obtained a PG transcriptome of ~49.5 Mb. A total of 191 PBE transcripts, which included pheromone biosynthesis activating neuropeptides, fatty acid transport proteins, acetyl-CoA carboxylases, fatty acid synthases, desaturases, β-oxidation enzymes, fatty acyl-CoA reductases (FARs) and fatty acetyltransferases (FATs), were selected from the dataset. A comparison of the E. cautella transcriptome data with three other Lepidoptera PG datasets revealed that 45 % of the sequences were shared. Phylogenetic trees were constructed for desaturases, FARs and FATs, and transcripts that clustered with the Δ14, Δ12 and Δ9 desaturases, PG-specific FARs and potential candidate FATs, respectively, were identified. Transcripts encoding putative pheromone degrading enzymes, and candidate pheromone carrier and receptor proteins expressed in the E. cautella PG, were also identified. Conclusions: Our study provides important background information on the enzymes involved in pheromone biosynthesis. This information will be useful for the in vitro production of E. cautella sex pheromones and may provide potential targets for disrupting the pheromone-based communication system of E. cautella to prevent infestations. Keywords: Ephestia, Pheromone, Pheromone gland, Transcriptome, Pheromone biosynthetic enzymes * Correspondence: [email protected] 1Department of Plant Protection, King Saud University, Chair of Date Palm Research, College of Food and Agricultural Sciences, Riyadh 11451, Saudi Arabia Full list of author information is available at the end of the article © 2015 Antony et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Antony et al. BMC Genomics (2015) 16:532 Page 2 of 26 Background by the brain, messages are then passed to the effector Pheromone-based methods of insect control are essential neurons, and finally the behavioral response is elicited. components of integrated pest management practices The expression of OR proteins is necessary and suffi- worldwide. The pheromones of over 2,000 insect species cient for odor detection in insects [25]. At first, volatile are now known, and The Pherobase is an updated compil- odors are bound to odorant-binding proteins (OBPs), a ation of pheromones and other behavior-modifying che- family that includes two sub-families, the pheromone- micals found in insects [1]. Common biosynthetic binding proteins (PBPs) and the general odorant-binding pathways have also been well-cited in many scientific pub- proteins (GOBPs) [26, 27]. Other important soluble se- lications over the last two decades, leading to production creted proteins that are found within the sensillum of species-specific pheromone compounds [2–5]. The fe- lymph include chemosensory proteins (CSPs) and the male pheromones of almost all moth species are multi- antennal binding protein X (ABPX) [28]. Finally, odorant component blends of long hydrocarbon chains (10 to 18 molecules bind with ORs located in the dendritic mem- carbons long), unbranched alcohols, and acetates or alde- brane of receptor neurons [27, 29]. Sensory neuron hydes, and are synthesized in the modified epidermal cells membrane proteins (SNMPs) are another class of proteins (pheromone-producing cells) from C16 or C18 fatty acid involved in pheromone reception at the olfactory receptor precursors [4, 6, 7]. A typical moth pheromone biosyn- neuron (ORN) [29–31]. Later, the signal termination is ac- thetic pathway begins even before the adult eclosion by complished by the odorant-degrading enzymes (ODEs, releasing pheromone biosynthesis activating neuropeptide also known as pheromone-degrading enzymes) [26, 32]. (PBAN) from the brain and transporting it to the phero- Knowledge of the olfactory communication system at the mone gland (PG), which in turn activates functional group molecular level in insects is still in its early stages. modification enzymes [3, 4, 8, 9] or acetyl-coenzyme A The tropical warehouse moth (almond moth), Ephestia (CoA) carboxylase (ACC) [10]. As the first step in cautella (Lepidoptera: Pyralidae) is a destructive pol- pheromone biosynthesis, carboxylation of acetyl-CoA yphagous storage pest of wheat flour, dried figs, dates, to malonyl-CoA is catalyzed by ACC [10]. This is nuts, chocolate, dried fruits, grain and associated proc- followed by fatty acid synthase (FAS) activity to pro- essed food products worldwide. The control of these duce saturated fatty acids (C18:0 and C16:0) using pests has depended exclusively on methyl bromide; how- malonyl-CoA as the substrate. Later, the fatty acyl desa- ever, methyl bromide was reported as facing an inter- turases (DESs) introduce double bonds in the acyl national phase-out by the year 2015 [33]. In this context, chains, and then, specific β-oxidation enzymes shorten pheromones hold great potential in insect pest manage- the chains. Once specific unsaturated pheromone pre- ment [34]. In the last few decades, the elucidation of cursors are formed, the terminal carboxyl group is pheromone biosynthetic pathways, and the molecular modified to form one of the functional groups, alcohol, al- characterization and functional gene expression of phero- dehyde or acetate ester (OH, CHO or OAc, respectively), mone biosynthesis enzymes (PBEs) and OR proteins in- and is catalyzed by fatty acyl reductase (FAR), aldehyde re- creased greatly [2, 10]. Most recently, through a synthetic ductase (AR) or fatty acetyltransferase (FAT), respectively biology approach, transgenic Nicotiana benthamiana [3–5, 10]. A variety of desaturases, which introduce double plants with insect desaturases, FARs and FATs produced bonds into the acyl at the Δ6 [11], Δ9[12–14], Δ10 [15], pure multi-component pheromone compounds [35]. Such Δ11 [13, 16, 17] and Δ14 [18] positions, have been cloned in vitro production technology (green technology) could and functionally expressed from many moth species be cost effective and produce isomerically pure com- [2–5, 10]. Great progress has also been made in the pounds that should be identical to chemically synthesized functional characterization of FARs since their discov- compounds. This research requires complete knowledge ery in Bombyx mori [19] through detailed studies of of the specific pheromone biosynthetic pathway and the pheromone evolution and the FARs of nine Ostrinia functional characterization of enzymes (genes) involved in spp. [20–22], Yponomeuta spp. [23], Helicoverpa spp. pheromone biosynthesis. and Heliothis spp.[24].However,themolecularchar- The rapid progress over the last decade resulted from the acterizations of other critical enzymes in the phero- convergence of modern techniques from different areas of mone biosynthetic pathway,suchasACC,FAS,and science has enriched our knowledge of the genetics of several β-oxidation and acetylation enzymes, have not pheromone-based communications and olfactory commu- been characterized at the enzymatic level in insects. nication systems. Transcriptome sequencing strategies are Female moths typically start releasing sex pheromones efficient for identifying